Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales

Abstract

Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescent proteins (FPs) engineered from bacterial phytochromes have become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.

Keywords: all-optical electrophysiology; bacterial phytochrome; biosensor; deep-tissue imaging; iRFP.

Figure 1.. NIR FPs and their applications in multiplexed imaging and optogenetics.

(A) Effective cellular brightness and emission wavelength maxima of NIR FPs. Early miRFPs and mIFP are shown as red circles, new miRFPs as blue circles, and dimeric iRFPs as larger green splitted circles. (B) Tubulin-miRFP720 localizes well in live HeLa cells. (C) miRFP670 and miRFP709 allow two-color labeling as shown by simultaneous visualization of H2B and vimentin in HeLa cells. (D, E) miRFPs and mIFP allow cross-talk free three-color imaging together with green and red GFP-like FPs as shown by structured illumination microscopy (SIM) in HeLa (D), and by visualization of abdomen muscle (mIFP), Class IV DA neurons (CD4-tdTomato), and extracellular collagen matrix (GFP) in Drosophila (E). C, D adapted under the Creative Commons Attribution license from [18]; E adapted with permission from [22], (F) miRFPs was used in four- color imaging to localize invadopod (marked by white arrow, miRFP703), colocolized with intracellular (EGFP) and extracellular (Alexa-565) MT1-matrix metalloproteinase (MMP), and matrix degradation (Alexa-405). Courtesy of Louis Hodgson (G-I) iRFP713 was used with blue-light induced optogenetic tools, including chanelrhodopsin ChR2 in muscle (G, adapted with permission from [33]), channel switch BACCS in neurons (H, adapted under the Creative Commons Attribution license from [34]) and system CRY2/CIBN for recruitment of inositol-5-phosphatase (5-ptase) to the cell membrane in COS-7 cells (I, adapted with permission from [35]). Scale bars, 5 μm (D, I), 10 μm (B, C, G), 20 μm (E, F), and 50 μm (H).

Figure 2.. Single-chain NIR biosensors for different targets in signaling cascades based on miRFP670-miRFP720 FRET pair.

(A) Schematics of the NIR FRET biosensors for AKAR PKA and JNKAR JNK kinases. FHA is the phosphopeptide binding domain that recognizes phosphorylated peptide marked as “subst“. (B,C) NIR AKAR PKA kinase biosensor was validated in live HeLa cell stimulated with 1 mM dibutyryl cAMP (dbcAMP). Time-lapse images (B) and the corresponding plot (C) are shown. (D) Schematics of the FRET Racl biosensor. PBD1 is a p21-binding domain 1, PBD2 is a mutant p21-binding domain. Racl is a full-length Racl post-translationally isoprenylated for membrane localization. (E) Combination of NIR and cyan-yellow FRET biosensors for simultaneous imaging of Racl and RhoA activities in a MEF/3T3 cell. DIC, differential interference contrast. Racl is predominantly localized at the leading edge, whereas RhoA activity is mostly localized at the retracting tail, the side edges and at the back of the leading-edge protrusions (see arrows). (B,E) FRET/donor ratio is shown in pseudocolor. Scale bar, 20 μm. (C) Mean ±s.e.m. (n=3) of the FRET/donor ratio for the whole cell plotted vs time. B,C,E adapted with permission from [19]

Figure 3.. Multiscale imaging of NIR reporters.

(A) Schematics of the IκBa-miRFP703 reporter for canonical activation of NF-κB pathway. Its activation by TNFα or LPS results in hcBa degradation together with miRFP703. (B,C) Visualization of IκBa-miRFP703 reporter in HEK293 cells and in a mouse liver. B,C adapted under the Creative Commons Attribution license from [18], (D) Schematics of the NIR cell cycle reporter. miRFP670-hGem(l/110) and miRFP709-hCdtl(l/100) are reciprocally produced and degraded in a cell cycle dependent manner. (E,F) Visualization of NIR cell cycle reporter in synchronized (in G2/M and G1 phases) and non-synchronized (mix) HeLa cells and in a mouse with implanted cells. E,F adapted under the Creative Commons Attribution license from [18], (G) Schematics of the NIR bimolecular fluorescence complementation reporters iSplit and miSplits. Two split fragments (the PAS and the GAF domains of iRFP or miRFP) are not fluorescent as separate polypeptides until two fusion proteins interact. Here FRB and FKBP interact in the presence of rapamycin (rapa). (H,I) Visualization of iSplit reporter in HeLa cells and in a mouse xenograft tumor. H,I adapted with permission from [47].

Figure 4.. High resolution in vivo imaging of NIR probes.

(A) iRFPs can be effectively excited by Ti-Sapphire laser at 880 nm as shown by two-photon (2P) absorption spectra of iRFP682 (open circles). One-photon (solid line) absorption spectra of iRFP682 is also shown. (B) Visualization of iRFP682 and EGFP in neurons of mouse cortex layer 2/3 using in vivo single-wavelength (880 nm) 2P microscopy. A,B adapted with permission from [10], (C) Visualization of iRFP713 in neurons of mouse cortex layers 1, 2/3, 4, 5 using 2P microscopy with adaptive optics wavefront correction. C adapted with permission from [9] and [56], (D) The photoacoustic tomography (PAT) of BphPl Pfr (marked as ON) and BphPl Pr (marked as OFF) allows to subtract background absorption by hemoglobin (Hb02) and deoxyhemoglobin (HbR). (E) BphPl can be repeatedly photoswitched by 780 and 630 nm light between its Pfr ON state and Pr OFF state. (F, G) Reversibly switchable photoacoustic computed tomography (RS-PACT) of deep-seated tumors in vivo. This technique can visualize kidney (F) and brain (G) tumors and vasculature as overlays of the ON state image (BphPl, pseudocolor) and the OFF state image (hemoglobin, gray) with exceptionally high resolution. F,G adapted with permission from [60], Scale bars, 100 μm (B), 20 μm (C).

Figure 5.. Optical technologies aided by NIR probes to address current challenges.

Challenging studies at different spatial scales from cells, cellular networks to organs and whole organisms (top, “Biological systems”) are presented in the “Current challenges” (middle). Corresponding optical technologies that rely on NIR FPs and NIR biosensors and could address the challenges are listed in the “Solutions” (bottom).